Intergenerational Effect Of Maternal Betaine On Hepatic Expression Of IGF2 In Rats And Its Mechanisms

Insulin-like growth factor 2(IGF2)is an important component of the insulin/IGF system and regulates cell growth,proliferation and metabolism.IGF2 in the blood is mainly derived from the liver.IGF2 is one of several hundred imprinted genes in mammals.Its transcriptional activity is not only affected by the methylation status of the promoter region but also by the methylation status of the imprinting control region(ICR).Betaine is a trimethyl derivative of glycine and is commonly found in human plasma due to dietary intake and endogenous synthesis in the liver and kidney.In the liver,its main role is as a methyl donor in metabolism.In the past,it has been found that the addition of betaine to diet can promote animal growth and may be associated with increased blood GH/IGF1 levels.The effect of betaine on the expression of IGF2 in animals has been rarely reported.However,in the past,studies on the growth-promoting of betaine have focused on the contemporary role or the maternal effect in the neonatal period.There are few reports on the effects of maternal betaine on the growth and development of F1 progeny after birth,and the research on the growth and development of F2 animals is rarely reported.The purpose of this study was to expound the intergenerational effect of maternal betaine on hepatic expression of IGF2 in rats,and to reveal its mechanism.1 Effect of maternal betaine supplementation on hepatic expression of IGF2 in F1 generation weaned rats and its mechanismsThree month old Sprague-Dawley females were used in this experiment,with an average body weight of 400 g purchased from the Experimental Animal Center of Jiangsu University.All rats were housed under controlled temperature(22 ± 0.5 ℃),humidity(50±5%)and light(12 hours light/12 hours dark)condition and animals were given free access to food and water.A total of 40 mating female rats(F0)were used for this experiment.The control group received a basal diet,while the betaine group was fed a diet supplemented with betaine.Betaine(purity 98%,Sigma-Aldrich)was mixed at a level of 10 g/kg in the basal diet.After the F1 generation of the pups were born(D0),recording the number of litters and the birth weight,then adjusting the number of litters each litter to 10(5 males and 5 females).F1 male offspring were weaned on day 21(D21),after which all basal diets were used in the two groups.Excepted to sampling,the remaining F1 pups with their littermates were raised to the 45th day of age(D45),and then 10 male pups(nearly the average body weight of their litter)were selected from each group and raised to 63 days old(D63)alone.Maternal betaine supplementation significantly increased the number of litters and litter weight(P<0.05).The F1 male rats in the betaine group had lower birth weight,weaning weight and 45-day-old body weight than the control(P<0.05).The average daily gain during the period from 45 days to 63 days in the betaine group was significantly higher than that in the control group(P<0.05),and there was no difference in body weight at 63 days.After immunohistochemistry staining,the liver proliferating cell nuclear antigen(PCNA)of the F1 weaned male rats in the betaine group was higher than that of the control group(P<0.05).The levels of PCNA protein,CCND1 and PCNA in liver tissue were significantly higher in the betaine group than in the control group(P<0.05),indicating an increase in hepatocyte proliferation.Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)results shown that the number of TUNEL positive cells in the betaine group was higher than that in the control group(P<0.05),indicating that hepatocyte apoptosis was also enhanced.Intracellular signaling pathway detection revealed that both ERK-mediated proliferation and AMPK-mediated apoptotic pathways in the betaine group were activated.Immunohistochemical staining showed that IGF2 in the liver of the betaine group,IGF2 protein level in the liver tissue and serum free IGF2 level in the weaning group were higher than the control group(P<0.05).The mRNA levels of four IGF2 variants in the betaine group were higher than those in the control group(P<0.05),and the ratio of total IGF2 to H19 mRNA was higher than that of the control group(P<0.05).Further studies showed that the methylation level of the IGF2 gene promoter region in the F1 generation weaned liver was unchanged in the betaine group,but three of the CTCF binding sites in the four IGF2/H19 imprinting control regions were significantly higher in the betaine group than in the control group(P<0.05).In addition,the protein levels of betaine homocysteine methyltransferase(BHMT)and DNA(cytosine-5)-methyltransferase 1(DNMT1)in betaine group were significantly higher than those in the control group(P<0.05).The above results suggest that the hepatic methylation process of F1 male rats in the betaine group is enhanced during weaning,which changes the methylation status of ICR.The hypermethylation status of ICR may be associated with elevated IGF2 transcription.On the other hand,the progeny of the maternal betaine group may contact the additional betaine by breast milk or feeding,and activated the apoptotic signaling pathway in liver.2 Effect of maternal betaine supplementation on hepatic expression of IGF2 in F1 generation adult rats and its mechanismsStudies on maternal betaine affacts the growth and metabolism of neonatal have been reported.However,studies on maternal betaine affacts during the adulthood of F1 generations have not been reported.The previous study found that the F1 offspring male rats in maternal betaine group had lower birth weight and weaning weight,but they showed catch-up growth in adulthood.To reveal this phenomenon,we further studied the proliferation and apoptosis of liver cells in male rats at D63,and the related mechanisms of hepatic IGF2 expression and its regulation.The number of PCNA positive nulcei in the liver of F1 adult male rats in the betaine group was higher than that in the control group(P<0.05).In the betaine group,the levels of PCNA protein,CCND1 mRNA and PCNA mRNA in liver were significantly higher than in the control group(P<0.05),indicating an increase in hepatocyte proliferation.However,the number of TUNEL positive nulcei in the betaine group was lower than control(P<0.05),indicating that the apoptosis of hepatocytes was weakened.Intracellular signaling pathway detection showed that AKT/ERK-mediated proliferation was activated in the betaine group but AMPK-mediated apoptosis pathway was attenuated.In the betaine group,liver IGF2 immunohistochemical staining,IGF2 protein levels and serum free IGF2 levels were higher than the control group(P<0.05)at D63.Similar to weaning,the mRNA levels of four IGF2 variants in the betaine group were higher than those in the control group(P<0.05),and the ratio of total IGF2 to H19 mRNA was higher than that of the control(P<0.05)at D63.The results of MeDIP shown,the methylation levels of the four IGF2 promoter regions in the liver were unchanged in the betaine group.However,three of the CTCF binding sites in the four IGF2/H19 imprinting control regions were significantly higher in the betaine group than control(P<0.05).There was no change in the levels of BHMT and DNMT1 related proteins in the liver of betaine group.From the above results,it is speculated that the elevated level of liver IGF2 transcription may be related to the hypermethylation status of ICR.From weaning to adulthood,both groups were fed a basal diet,suggesting that the ICR methylation imprint in adulthood may be retained from weaning to adulthood.3 Effect of maternal betaine supplementation on hepatic expression of IGF2 in F2 generation weaned rats and its mechanismsIn the previous study,we investigated the effects of maternal betaine on the growth of F1 rats and the hepatic expression of IGF2,but no reports on the affects of F2 rats have been reported.In order to understand the effects of maternal betaine on the growth of rat and IGF2 expression in the liver,we observed the growth of F2 male rats at weaning,and further studied the proliferation and apoptosis of F2 generation weaned liver cells.And related mechanisms in the regulation of IGF2 expression.A total of 40 mated female F1 rats were used for this experiment.According to different sources of female rats,they were divided into F2 control group and F2 betaine group,with 20 rats in each group.After the F2 generation of the pups were born(D0),recording the number of litters and the birth weight,then adjusting the number of litters per litter to 10(5 males and 5 females).F2 male offspring were weaned and sampled on day 21(D21).The addition of betaine to the ancestral mother’s diet did not affect the number of litters and litter weight of the F2 generation.There was no difference in the birth weight of the F2 male rats in the betaine group compared with the control group.However,F2 weaned males weighed more than the control(P<0.05).The number of PCNA positive nuclei in the liver of F2 weaned male rats in the betaine group was higher than that in control(P<0.05).The levels of PCNA protein,CCND1 mRNA and PCNA mRNA were higher in betaine group than control(P<0.05),indicating an increase in hepatocyte proliferation.However,the number of Tunel positive nuclei in the liver of the betaine group was lower than that of the control group(P<0.05),indicating that the apoptosis of hepatocytes was weakened.Intracellular signaling pathway detection revealed that AKT/ERK-mediated proliferation was activated but the AMPK-mediated apoptotic pathway was attenuated in the betaine group.In F2 weaned male rats in betaine group,IGF2 immunohistochemical staining,IGF2 protein levels and serum free IGF2 levels were higher than those in the control group(P<0.05).The total IGF2 and H19 mRNA levels in betaine group were higher than control group(P<0.05),but the ratio of total IGF2 to H19 mRNA was unchanged.All of 4 IGF2 transcript variants mRNA in betaine group were higher than control(P<0.05).MeDIP results shown that the methylation of CTCF binding sites in 4 IGF2/H19 imprinting control regions was not changed in the two groups during F2 generation weaning,but the methylation levels of the four different IGF2 promoter regions were lower in the betaine group than in the control group(P<0.05).The protein associated with the methylation process was only DNA(cytosine-5)-methyltransferase 3A expression reduced in the betaine group(P<0.05).The above results indicated that the addition of betaine to the ancestral mother’s diet affect the IGF2 transcript level in the F2 generation of weaned rats,along with reducing the methylation of IGF2 promoter,rather than ICR.4 Effect of betaine on the expression of IGF2 gene in HepG2 cells and its mechanismsIn the previous in vivo experiments,the maternal betaine promoted IGF2 gene expression in the offspring liver at both weaning and adult and altered the methylation modification of the IGF2/H19 imprinting control region.In order to test whether the addition of betaine can directly affect the IGF2 gene expression of hepatocytes,whether the methylation status of the imprinted control region is also changed,and whether it can be transmitted to the progeny cells through mitosis.We using betaine to treat HepG2 cells as a model performed the following study.HepG2 cells were cultured using complete medium(high glucose DMEM medium containing 10%FBS,antibiotics).In each well of the six-well plate were seeded 5×105 cells.After 48 h,the cells were treated with betaine and treated for 24 h(P0 cells).P0 cells were digested and plated for 48 h,and then harvested for 24 h without addition of betaine(P1 cells).After treatment of HepG2 cells with different concentrations of betaine,it was found that the CCK8 activity of P0 generation HepG2 cells was significantly higher than that of the control after 48 h at 20 mM final concentration(P<0.05).Based on the results of CCK8,a 20 mM betaine concentration was selected for subsequent experiments.The activity of CCK8 in P1 generation HepG2 cells were significantly higher than that of the control at 48 h and 72 h(P<0.05).Cell cycle analysis by flow cytometry revealed that the proliferation of P0 and P1 cells was higher in the betaine group than in the control group(P<0.05).Similarly,Western Blot assay showed that the PCNA protein levels of P0 and P1 cells in the betaine group were higher than those in the control group(P<0.05).In the betaine group,the Caspase 3 activity of P0 cells was higher than that of the control(P<0.05),but the Caspase 3 activity of P1 cells was lower than that of the control group(P<0.05).Similarly,the results of apoptosis using flow cytometry showed that the apoptosis level of P0 cells in the betaine group was higher than that in the control group(P<0.05),but the P1 cells were lower than the control group(P<0.05).Intracellular signaling pathway detection revealed that both AKT/ERK-mediated proliferation and AMPK-mediated apoptotic pathways were activated in the P0 generation cells.In the P1 generation cells,AKT/ERK-mediated proliferation was activated in the betaine group,but the AMPK-mediated apoptotic pathway was attenuated.Similar to the weaning and adult experiment in the F1 generation of rats,the IGF2 protein levels in both P0 and P1 cells of the betaine group were higher than those of the control(P<0.05).The ratio of IGF2 to H19 mRNA was increased at the transcriptional level(P<0.05),and the mRNA levels of the four IGF2 transcript variants were also increased in P0 and P1 of the betaine-treated group(P<0.05).MeDIP results shown that the methylation levels of the four IGF2 promoter regions were unchanged in the P0 and P1 cells of the betaine group,but the methylation level of the CTCF binding sites in the IGF2/H19 ICR was increased(P<0.05).Detection of methionine cycle-related proteins revealed that betaine increased glycine N-methyltransferase in P0 cells(P<0.05).There was no change in the methionine cycle-related protein and DNMT1 in the P1 cells of the two groups.The above results indicate that betaine can increase IGF2 expression in HepG2 cells.And the hypermethylated CTCF binding sites of the imprinted control region can be delivered from P0 to P1 cells.In summary,maternal betaine increased the expression of IGF2 in the liver of the F1 progeny during weaning and adulthood,accompanied by hypermethylation of the IGF2/H19 imprinting control region,and increased IGF2 expression in rat liver during weaning of F2 progeny was accompanied by hypomethylation of the IGF2 promoter.In vitro,betaine can alter the methylation status of the IGF2/H19 imprinting control region of HepG2 cells and pass it to progeny cells.